Effect of Increased Blood Levels of β-Endorphin on Perception of BreathlessnessEffect of 03B2-Endorphin on BreathlessnessFREE TO VIEW

From the Section of Pulmonary and Critical Care Medicine (Drs Mahler and Gifford) and the Pulmonary Function and Cardiopulmonary Exercise Laboratories (Ms Waterman and Mr Ward), Dartmouth-Hitchcock Medical Center, Lebanon, NH; the Department of Kinesiology and the Department of Physiology and Neurobiology (Drs Kraemer and Kupchak), University of Connecticut, Storrs, CT; and the Department of Public Health Sciences (Dr Harver), University of North Carolina at Charlotte, Charlotte, NC.

Abstract

Background:Although opioid receptors are expressed broadly in the CNS and in peripheral sensory nerve endings including bronchioles and alveolar walls of the respiratory tract, it is unknown whether the modulatory effect of endogenous opioids on breathlessness occurs in the CNS or in the peripheral nervous system. The purpose of this investigation was to examine whether increased blood levels of β-endorphin modify breathlessness by a putative effect of binding to peripheral opioid receptors in the respiratory tract.

Methods:Twenty patients with COPD (10 women and 10 men; age, 70 ± 8 years) inspired through resistances during practice sessions to identify an individualized target load that caused ratings of intensity and unpleasantness of breathlessness ≥ 50 mm on a 100-mm visual analog scale. At two interventions, blood levels of β-endorphin and adrenocorticotropic hormone (ACTH) were measured, ketoconazole (600 mg) or placebo was administered orally, and patients rated the two dimensions of breathlessness each minute during resistive load breathing (RLB).

Results:By inhibiting cortisol synthesis, ketoconazole led to significant increases in β-endorphin (mean change, 20% ± 4%) and ACTH (mean change, 21% ± 4%) compared with placebo. The intensity and unpleasantness ratings of breathlessness and the endurance time during RLB were similar in the two interventions.

Conclusions:The previously demonstrated modulatory effect of endogenous opioids on breathlessness appears to be mediated by binding to receptors within the CNS rather than to peripheral opioid receptors in the respiratory tract. An alternative explanation is that the magnitude of the β-endorphin response is inadequate to affect peripheral opioid receptors.

Figures in this Article

Opioid peptides and their receptors are expressed broadly throughout the CNS and the peripheral nervous system, including the respiratory tract.1,2 β-Endorphin, an endogenous opioid processed from the cleavage of proopiomelanocortin (POMC), is released from the pituitary gland into the circulation and from hypothalamic neurons into cerebrospinal fluid and the brain in response to stress and noxious stimuli.3‐6 Multiple types of opioid receptors are expressed broadly in the cortex, limbic system, and brain stem of the CNS and in peripheral sensory nerve endings including the bronchioles and alveolar walls of the respiratory tract (epithelial and/or pulmonary C fibers).1,2,7,8 Using naloxone to block both peripheral and central opioid signaling, we demonstrated that endogenous opioids modulate the perception of breathlessness during exercise and resistive load breathing (RLB) in patients with COPD.9,10 However, it was not possible to determine whether endogenous opioids modulate breathlessness by binding to opioid receptors within the CNS and/or at peripheral nerve endings.

The purpose of this investigation was to examine whether increased blood levels of β-endorphin modify breathlessness by a putative effect of binding to peripheral opioid receptors in the respiratory tract. Although human β-endorphin has been administered via IV to investigate its analgesic effect in healthy volunteers and patients,11,12 we chose an alternative approach because there is no standard dose of IV β-endorphin for any specific indication and we were concerned about the potential adverse effects of administering a protein via IV, including infusion or allergic reactions, relative to the theoretical benefits on dyspnea perception. To augment the levels of circulating β-endorphin, we administered ketoconazole, an oral antifungal antibiotic that inhibits cortisol synthesis. This inhibition activates the hypothalamic-pituitary-adrenal axis such that corticotrophin-releasing hormone is secreted from the hypothalamus, which stimulates the release of POMC. POMC-derived peptides are cleaved, leading to secretion of β-endorphin and adrenocortiocotropic hormone (ACTH) into the circulation.13,14 Based on this established mechanism of action, there would be no expected release of endogenous opioids from hypothalamic neurons into the CNS with ketoconazole. Furthermore, circulating endogenous opioids do not cross the blood-brain barrier and enter the CNS.3,8 Jarmukli and colleagues15 used this approach to demonstrate that ketoconazole, at a dose of 1,800 mg, increased the circulating levels of β-endorphin compared with baseline values in eight male patients with coronary artery disease. The release of β-endorphin was associated with an increase in the threshold for peripheral pain induced by a radiant heat source but it did not alter the angina threshold of these patients during exercise, compared with placebo.15

The null hypothesis of our study was that there is no difference between ratings of the intensity and/or unpleasantness of breathlessness during RLB with ketoconazole compared with placebo. First, an open-label trial was performed to establish that oral ketoconazole increased circulating concentrations of β-endorphin in patients with COPD. Subsequently, a randomized, placebo-controlled, double-blind trial was performed to investigate the effect of increased levels of circulating β-endorphin on the perception of breathlessness.

A secondary purpose was to explore whether increases in circulating levels of β-endorphin with ketoconazole or the development of dyspnea during RLB was associated with changes in the blood levels of substance P. Substance P is an excitatory neurotransmitter released from sensory nerve fibers that transmits pain information into the CNS.16 Substance P and its endogenous receptor, neurokinin 1, are also found in airway smooth muscle, blood vessels, mucous glands, immune cells, and sensory nerves.17 Elevated levels of this neuropeptide have been identified in induced sputum from patients with asthma and chronic bronchitis.18

Materials and Methods

Subjects

Patients with a diagnosis of COPD were recruited from the out-patient clinic at our institution and provided informed written consent. Each patient reported breathing difficulty with activities of daily living. Inclusion criteria were age ≥ 50 years, a diagnosis of COPD,19 at least a 10 pack-year history of smoking, postbronchodilator FEV1 30% to 80% predicted, and postbronchodilator FEV1/FVC < 70%. Exclusion criteria were current smoking, current use of narcotic medications, and current use of a reported drug that could cause a possible drug interaction with ketoconazole. Patients continued usual COPD medications throughout the study.

Study Designs

Each study protocol was approved by the Committee for the Protection of Human Subjects at Dartmouth College (CPHS No. 22431 and No. 22775). Study 1 was open label and included two visits (2-3 days apart). Study 2 was a randomized, cross-over, placebo-controlled, double-blind trial with three visits (2-3 days apart). Study medications were prepared by the research pharmacy at our institution.

Interventions

Study 1:

Study 2:

Three 200-mg capsules of ketoconazole (600 mg) or three capsules of inert powder (with appearance identical to ketoconazole) were administered orally in random order at 8:30 am.

Procedures

Study 1:

At visit 1, baseline data were collected, and each patient was familiarized with the study protocol. Spirometry (Nspire model HD3000) was performed using standard techniques.20 The sequence of testing at visit 2 is summarized in Table 1.

5. Three and one-half hours after taking the study medication, the patient performed spirometry before and 20 min after inhaling two puffs of albuterol (180 μg) to provide standardized bronchodilation prior to RLB.

6. Four hours after taking ketoconazole, 10 mL of venous blood was removed by venipuncture.

7. Patient breathed through a mouthpiece for 5 min with no resistance, and then the target resistance load (determined at visit 1) was applied and patient breathed for “as long as possible.”

8. Each minute patient rated the intensity and unpleasantness of breathlessness on a separate visual analog scale.

9. RLB was terminated when patient stopped voluntarily or at 20 min.

10. Ten milliliters of venous blood was removed.

RLB = resistive load breathing.

Study 2:

At visit 1, patients rated their breathlessness on the self-administered computerized version of the baseline dyspnea index,21 and spirometry and diffusing capacity (Collins model CPL) were measured using standard techniques22,23 and expressed as percentages of predicted normal values.22,24 Breathlessness was defined for each patient as breathing difficulty. Patients were familiarized with rating breathlessness on separate 100-mm visual analog scales and performed RLB practice sessions to identify a target resistance that corresponded to mean ratings of ≥ 50 mm for either intensity or unpleasantness of breathlessness over 10 min.10 The sequence of testing at intervention visits 2 and 3 is described in Table 1.

Statistical Analysis

Ratings of the intensity and unpleasantness of breathlessness at equivalent times for each patient during RLB were primary outcomes.10 For example, if one patient provided 10 ratings during 10 min of RLB with ketoconazole and six ratings during 6 min of RLB with placebo, then ratings for intensity and unpleasantness through 6 min were used for analysis for that patient (ie, multiple isotimes). A secondary outcome was levels of β-endorphin and ACTH, whereas an exploratory outcome was levels of substance P. A paired t test was used to compare differences in outcomes between ketoconazole and placebo. A repeated-measures analysis of variance with Bonferroni correction for multiple comparisons was used to test for differences in blood substances with each intervention. Subgroup analysis for the primary outcome was planned a priori based on β-endorphin levels. A P value < .05 was considered statistically significant. Values are presented as mean ± SD.

A sample size of 20 was adequate to provide 80% power to detect a significant difference in breathlessness ratings (α = 0.05).9,28 Because no data are available on the impact of β-endorphin responsiveness to ketoconazole on breathlessness ratings, subgroup analysis of the primary analysis was considered exploratory.

Results

Study 1

Characteristics of the four female and four male patients are summarized in Table 2. As shown in Figure 1, all patients exhibited an increase in β-endorphin levels 4 h after administration of 600 mg of ketoconazole (142.4 ± 69.5 pg/mL) that was significantly greater than baseline (56.7 ± 30.1 pg/mL) (P = .008). There was no additional change in levels following the second dose of 600 mg of ketoconazole (143.8 ± 75.7 pg/mL) (P > .05). No patients reported any side effects with ketoconazole.

Study 2

Enrollment, allocation, follow-up, and analysis of patients are shown in Figure 2. Twenty of 23 patients who signed the consent form completed the study. One patient did not meet the inclusion/exclusion criteria, and no testing was performed. At visit 2, one patient could not tolerate breathing through the initial inspiratory resistance (15 cm H2O/L/s) and withdrew from the study; another patient gave ratings of breathlessness of < 50 mm on the VAS for the highest resistance (50 cm H2O/L/s) and was withdrawn from the study.

Descriptive characteristics of the 10 female and 10 male patients are displayed in Table 3. No differences were noted in postbronchodilator values of FEV1, FVC, or Spo2 between visits 2 and 3 (P > .05 for each comparison). The target inspiratory resistance was 36.3 ± 12.1 cm H2O/L/s (range, 15-50 cm H2O/L/s). There were no significant differences in physiologic variables (oxygen consumption, CO2 production, minute ventilation, respiratory rate, tidal volume, partial pressure of end-tidal CO2, Spo2, or heart rate) at rest or at the end of RLB between ketoconazole and placebo. As per study protocol, the RLB trial was stopped in four patients because endurance time was 20 min.

Patients provided a total of 252 ratings for each dimension of breathlessness at equivalent times during RLB. Average ratings for intensity (77.6 ± 22.3 mm vs 78.2 ± 21.4 mm; P = .45) and unpleasantness (72.5 ± 23.8 mm vs 72.6 ± 23.5 mm; P = .85) of breathlessness were similar for ketoconazole and placebo. Patients generally rated intensity higher than unpleasantness of breathlessness during RLB under both conditions (P < .001 for ketoconazole and for placebo). No difference was noted in endurance times during RLB between ketoconazole (14.0 ± 5.7 min) and placebo (14.2 ± 6.1 min) (P = .91).

Levels of serum β-endorphin, ACTH, and substance P in 18 patients are shown in Table 4. In two patients, blood could not be drawn for testing. Baseline levels of these substances were similar in the ketoconazole and placebo groups. Significant increases were identified in concentrations of β-endorphin and ACTH with ketoconazole at 4 h and after completion of RLB with ketoconazole, but not with placebo (Fig 3). Substance P increased similarly at 4 h with both interventions; after RLB there was a significant increase in substance P with ketoconazole, but not with placebo (Fig 4).

Figure Jump LinkFigure 4.Box plot of values (pg/mL) for substance P in 18 patients with COPD. In two of the 20 patients, blood could not be drawn. Substance P levels with Keto and placebo were compared. *P < .0001 at end-RLB. See Figure 1 and 3 legends for expansion of other abbreviations.Grahic Jump Location

Discussion

The unique findings of this study in patients with COPD were (1) ketoconazole led to significant increases in circulating levels of β-endorphin and ACTH compared with placebo; (2) there were no differences in patient ratings of intensity and unpleasantness of breathlessness during RLB between ketoconazole and placebo; and (3) substance P increased over 4 h with both treatments, whereas a further increase was observed after RLB only with ketoconazole.

We administered oral ketoconazole to pharmacologically augment circulating levels of β-endorphin to investigate whether endogenous opioids act on receptors in the respiratory tract to modify breathlessness. As a potent inhibitor of cortisol production, ketoconazole causes the release of corticotropin-releasing hormone from the hypothalamus to stimulate the release of β-endorphin and ACTH from the anterior pituitary gland into the circulation.13,14 The comparable increases in levels of both β-endorphin (mean ∆ = 20% ± 4%) and ACTH (mean ∆ = 21% ± 4%) at 4 h after ketoconazole administration in the patients reflect activation of the hypothalamic-pituitary-adrenal axis. Circulating endogenous opioids do not cross the blood-brain barrier,3,8 and only a negligible amount of ketoconazole, even at doses > 600 mg, is evident in cerebral spinal fluid.30 In a review of the medical literature, we could not find any evidence that ketoconazole has a direct effect on hypothalamic neurons that causes the release of β-endorphin into the CNS, or that ACTH affects the perception of breathlessness.

The 600-mg dose of ketoconazole administered 4 h prior to RLB was selected based on our findings in an open-label study of eight patients with COPD (study 1). Four hours after an initial dose of 600 mg of ketoconazole, there was a significant increase in β-endorphin compared with baseline, although individual variability was noted (Fig 1). There was no further augmentation in levels of circulating β-endorphin after a second 600-mg dose. In study 2, we observed a significant increase in β-endorphin (mean ∆ = 20% ± 4%) at 4 h in response to ketoconazole compared with placebo (mean ∆ = 4% ± 4%) (P = .002), although individual variability was evident (Fig 3). Based on our concerns about patient safety and compliance, we did not replicate the administration of ketoconazole to patients with coronary artery disease (patients took 1,200 mg at midnight and 600 mg at 6 am unsupervised at their home) as used by Jarmukli and colleagues.15

There were no differences in ratings of the intensity and unpleasantness of breathlessness during RLB associated with the increase in circulating β-endorphin with ketoconazole. In addition, comparison of ratings of breathlessness between the nine subjects with the greatest percentage increase (mean ∆ = 35% ± 6%) in β-endorphin response with ketoconazole and the nine subjects with the smallest percentage increase (mean ∆ = 7% ± 4%) showed no difference (Table 5). We concluded that the most likely explanation is that circulating β-endorphin does not act on peripheral opioid receptors located in the respiratory tract to modulate either the sensory (intensity) or affective (unpleasantness) dimensions of breathlessness. These findings are consistent with the results of Jarmukli and colleagues,15 who found that increased levels of β-endorphin had no effect on angina threshold during exercise in patients with coronary artery disease. In addition, randomized clinical trials have demonstrated that nebulized opioids, which would be expected to act on opioid receptors in the respiratory tract, were no better than nebulized saline for relieving dyspnea.31‐33 Our results suggest that the modulatory effect of β-endorphin on breathlessness occurs within the CNS.

Based on the current understanding of other sensory experiences such as pain, we believe that other neuropeptides, in addition to endogenous opioids, likely contribute to and/or modulate the perception of breathlessness. For example, both endogenous opioids and their receptors, as well as substance P and its receptor, neurokinin 1, are distributed in the peripheral nervous system and the CNS.34 Furthermore, both of these neuromodulatory systems are present in the lung.2,35 We found that substance P levels increased over 4 h (from about 8:15 am to about 12:15 pm) with both ketoconazole and placebo, suggesting that this change may be a natural process. After the stimulus of RLB, substance P levels increased significantly with previous administration of ketoconazole (mean ∆ = 44% ± 12%) compared with placebo (mean ∆ = 4% ± 7%) (P < .0001). At the cellular level, the perception of pain is related to the release of substance P from the peripheral terminals of sensory nerve fibers, whereas endorphins are released simultaneously from the dorsal horn to bind to opioid receptors and attenuate nociceptive neurons.6 Whether a similar interaction occurs with the opposing effects of substance P (excitatory) and β-endorphin (inhibitory) in the perception of dyspnea is an interesting, but unknown, consideration.

Our study had some limitations. First, in study 2 we observed a mean increase of 20% ± 4% in levels of β-endorphin measured at 4 h after administration of ketoconazole. It is possible that the magnitude of this response was inadequate to affect the peripheral opioid receptors and alter the perception of dyspnea. However, subgroup analysis revealed no trend or significant difference in ratings of breathlessness between patients with higher and lower β-endorphin responses. Whether a different dose or dosing schedule of ketoconazole would have changed our results is unknown. Second, there are no data on whether endogenous opioids are released into the CNS with ketoconazole. However, if ketoconazole caused the release of β-endorphin into the CNS, we would expect that such a response would decrease patient ratings of breathlessness. Third, we cannot exclude a type 2 error based on our sample size of 20 participants. Fourth, although RLB is an established laboratory method for studying mechanisms contributing to dyspnea, patient ratings of breathlessness during RLB may not relate to the experience of patients performing activities of daily living.36

Conclusions

In conclusion, to the best of our knowledge, our study is the first to examine whether a specific endogenous opioid, β-endorphin, modulates breathlessness by binding to receptors within the respiratory tract. Our results support the concept that endogenous opioids, and most likely exogenous opioids, act as neuromodulators of the perception of breathlessness within the CNS rather than at peripheral opioid receptors. An alternative explanation is that the magnitude of the β-endorphin response in our study was inadequate to affect the peripheral opioid receptors. Our data are consistent with previous findings that increased blood levels of β-endorphin did not modulate angina pectoris in patients with coronary artery disease.15 Whether substance P plays a role in the perception of breathlessness requires further investigation.

Acknowledgments

Author contributions: Dr Mahler is the guarantor of the manuscript and takes responsibility for the integrity of the data and accuracy of the data analysis.

Dr Mahler: contributed to all aspects of the investigation, including the final content of the manuscript, and as the principal investigator.

Dr Gifford: contributed to the design of the study, statistical analysis, and review of the manuscript.

Ms Waterman: contributed to the data collection, statistical analysis, and review of the manuscript.

Mr Ward: contributed to the design of the study, data collection, and review of the manuscript.

Dr Kraemer: contributed to the design of the study, measurement of blood levels, and review of the manuscript.

Dr Kupchak: contributed to the design of the study, measurement of blood levels, and review of the manuscript.

Dr Harver: contributed to the design of the study and review of the manuscript.

Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsor had no role in the conduct of the study, data analysis, manuscript preparation, or manuscript review.

Figure Jump LinkFigure 4.Box plot of values (pg/mL) for substance P in 18 patients with COPD. In two of the 20 patients, blood could not be drawn. Substance P levels with Keto and placebo were compared. *P < .0001 at end-RLB. See Figure 1 and 3 legends for expansion of other abbreviations.Grahic Jump Location

5. Three and one-half hours after taking the study medication, the patient performed spirometry before and 20 min after inhaling two puffs of albuterol (180 μg) to provide standardized bronchodilation prior to RLB.

6. Four hours after taking ketoconazole, 10 mL of venous blood was removed by venipuncture.

7. Patient breathed through a mouthpiece for 5 min with no resistance, and then the target resistance load (determined at visit 1) was applied and patient breathed for “as long as possible.”

8. Each minute patient rated the intensity and unpleasantness of breathlessness on a separate visual analog scale.

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